O-11: AUTOMATED QUANTIFICATION OF POCKED RED BLOOD CELLS CORRELATES WITH SPLEEN SIZE IN SICKLE CELL DISEASE
Authors: SISSOKO A., FRICOT A., ROUSSEL C., MANCEAU S., DUMAS L., CAPITO C., ALLALI S., YEKKACHE N., DUSSIOT M., NGUYEN Y., LEFORT A., AUSSILHOU B., TICHIT M., HARDY D., MAÎTRE B., MICHEL A., DE MONTALEMBERT M., CAVAZZANA M., JOSEPH L., BUFFET P.
Journal: HemaSphere (2022)

Purpose: The spleen is a lymphoid organ that protects the body against blood infections and filters blood cells. Defective spleen function (hyposplenism) is caused by splenectomy or by immunological or hematological diseases. Counting vacuole-containing RBC (pocked RBC) by Differential Interference Contrast microscopy (DIC) is a robust marker of defective splenic function but it relies on the expertise of fewer and fewer specialized laboratory technicians, hence its rare application in routine medical use. Materials and methods: We developed an automated method using a common fluorescent cytoplasmic marker, and imaging flow cytometry to quantify pocked RBC in 128 adult patients with Sickle Cell Disease (SCD). Spleen size was determined by 2 members of the team and scored as 4 categories: Splenomegaly, Normal, Small and Atrophic. Six children with SCD who had partial splenectomy for hypersplenism were also included. Spleen were retrieved immediately after surgery, histology and electronic microscopy were performed. A venous blood sample was collected at the time of surgery and the splenic blood was retrieved from spleen fragments. Results: The proportion of RBC containing one or more fluorescent dots by IFC correlated with results of the conventional method, i.e., the operator-dependent quantification of pocked RBC by differential interference contrast microscopy. Proportions of pocked RBC in circulation correlated with spleen size, which varied widely from splenomegaly to atrophic spleen. Histology of post-splenectomy samples from 3 SCD children with normal, moderately, or markedly elevated pocked RBC counts, showed either an almost normal aspect with congestion, intense sickling, or widespread fibrosis, respectively. Electronic microscopy of the spleen from the child with normal pocked RBC counts showed persistent RBC filtration through inter-endothelial slits. Intrasplenic blood was enriched in RBC displaying a peripheral fluorescent dot, highly suggestive of ongoing intrasplenic expulsion of vacuoles (pitting). Operator-independent, fluorescent-based quantification of pocked RBC correlates with the reference method and confirms that spleen function is partially preserved in many adults with SCD. Conclusion: Because quantification of pocked-RBC correlates with the results of splenic scintigraphy, the new fluorescence-based counting method is a solid operator-independent quantitative surrogate of spleen function, is simple, and can be performed within hours on less than 20 µl of venous blood. Some experts consider that adults with SCD are almost constantly asplenic with an atrophic spleen. In our study, patients with either splenomegaly, a normally-sized or small spleen had more variable counts, suggesting that spleen filtering function is often preserved in SCD, at least partially, even in adulthood. In summary, this new fluorescence-based quantification of pocked-RBC, based on the spontaneous accumulation of the dye in vacuoles, correlates with the reference method, opening the way to a robust, operator-independent quantification of spleen function. We show that spleen function is partially preserved in many adults with SCD. We also show that the fluorescent vacuoles accumulating in RBC from SCD patients are indeed expelled by the spleen-specific pitting process, as long as sinusal structures persist in the partially damaged SCD spleens. M. CAVAZZANA declares a conflict of interest: Consultancy, Expert: consultant for Smart-Immune biotech